Project Summary/Abstract ? Project 2 In people with obstructive sleep apnea (OSA), airflow obstruction results in hypercarbia and other signals that increase ventilation, dilate the airway, and also trigger cortical arousals from sleep. Current therapies such as CPAP focus on airway opening, but compliance with these therapies is poor, and many patients continue to have daytime sleepiness. As recurrent arousals from sleep contribute to daytime sleepiness and other consequences of OSA, new methods that maintain sleep in OSA without disrupting ventilation would address an important, unmet need in OSA treatment. In the last cycle of this P01, Dr. Saper?s group (Project 1) showed that calcitonin gene-related peptide (CGRP) neurons of the lateral parabrachial nucleus are necessary for cortical arousals in response to hypercapnia. Specifically, inactivation of PBCGRP neurons substantially delays or eliminates cortical arousals in response to hypercapnia without blunting ventilatory responses. Thus, the PBCGRP neurons are essential for driving cortical arousals, but they are not necessary for ventilatory responses to hypercapnia. We hypothesize that activation of inhibitory inputs to the PBCGRP neurons will delay or eliminate cortical arousals to hypercapnia without altering ventilatory responses. Our Aims seek to identify these inputs and their receptors on the PBCGRP neurons, with the ultimate goal of selectively reducing activity in the PBCGRP neurons to prevent cortical arousals while preserving ventilatory responses. This Project synergizes well with Projects 1, 3, and 4 that seek to enhance ventilatory responses to hypercapnia in mice, and Project 5 which seeks to identify pharmacological methods to improve OSA in people. We will first use conditional and conventional tracing methods to identify afferents to the PBCGRP neurons, and then we will use Channelrhodopsin-assisted circuit mapping (CRACM) to establish synaptic connectivity. Using single cell sequencing techniques, we will then identify receptors expressed by the PBCGRP neurons, and confirm receptor expression using in situ hybridization and in vitro calcium imaging. We will then use fos and fiber photometry to determine which afferent pathways to the PBCGRP neurons are sleep-active. Last, we will determine whether signaling through inhibitory inputs to the PBCGRP neurons delays or eliminates cortical arousals triggered by brief period of hypercapnia. We will measure the latency to cortical arousal after hypercapnia in combination with photostimulation of inhibitory inputs to the PBCGRP neurons and then with pharmacological inhibition of the PBCGRP neurons. Collectively, these multidisciplinary experiments will identify crucial anatomical and neurochemical inputs to the PBCGRP neurons that should provide new pharmacological opportunities for maintaining sleep in OSA without inhibiting airway opening.